The preparation of polyacrylamide-based matrices for electrophoresis has conventionally involved the aqueous copolymerisation of acrylamide with a crosslinking agent by free radical chemistry. The free radical polymerisation can be initiated by various processes, and once commenced, the polymerisation reaction proceeds until a gel is formed. Gels are often prepared on an individual basis prior to use, and there can be variations between gels that have been cast separately such that comparison between separations using the same gel type are not reliable. Additionally, there has now been a move to the commercial preparation of preformed gels which should have consistent quality and stable physical characteristics between batches.
Traditionally, polyacrylamide gels have been prepared in glass supports. For commercialisation purposes, synthetic electrophoresis gel supports offer a number of advantages over the traditional supports. These include versatility in processing, light weight properties, improved visual appearance, and shatter resistance.
It has long been recognised that the polymerisation and copolymerisation of acrylamide by free radical chemistry is subject to inhibition by a range of compounds. Specifically, oxygen acts to terminate growing polymer chains resulting in longer polymerisation times. Work by other commercial corporations (Daiichi Pure Chemicals and Novel Experimental Technologies) has recognised the impact of such inhibitors and have attempted to address the issues associated with them.
U.S. Pat. No. 5,350,552 (Daiichi Pure Chemicals) describe a batch process in which polyacrylamide gels for electrophoresis are prepared in a container with a low oxygen atmosphere. The batch process involves placing gel supporting plates into the container, in which they are separated by the aid of partition members. The purpose of the partition members is to act as “polymerisation prevention plates” to assist in enhancing and speeding up the cleaning and processing of the gels with a minimum of gel rejection. The partition members are also used to dissipate the polymerisation exotherm, which is thought to ultimately limit the batch size (up to a maximum of 50 Evalu cassettes in the batch). The partition members may be made from a variety of synthetic materials such as polyolefins, polystyrene or fluorinated resins, or from rubber, but should be able to “embrace a large amount of oxygen around its surface with high radical absorptivity.” The gel support plates themselves may be made out of glass or plastic.
In U.S. Pat. No. 5,350,552, the inventors recognised the requirement of preparing the gels in a low oxygen environment in order to eliminate the appearance of “flaws or stripes” in the gel. The minimisation of flaws in the gel has traditionally been achieved by the use of an overlay solution, which is employed to prevent the re-absorption of oxygen from the atmosphere by the top portion of the gel. The use of such conditions emphasise that oxygen in the container is a problem, and removal of the oxygen is possible either through application of a vacuum or by displacement with an oxygen-free gas such as nitrogen. After the container is filled with nitrogen, the gel solution is introduced. However, it is not specified whether nitrogen flow is maintained during the polymerisation.
U.S. Pat. No. 5,685,967 (Novel Experimental Technologies) describes a process by which a mould for an electrophoresis gel is coated with barrier films, such as silicone oxide, in order to form a polyacrylamide gel suitable for biological separations. Examples are given in the body of this patent in which various plastic materials, coated and uncoated, were examined for their influence on the gel polymerisation, the resultant physical properties and separation. As an illustration, uncoated SAN (styrene-acrylonitrile) cassettes induced poor polymerisation, and correspondingly, poor electrophoresis results. When the SAN material was coated with PET-SiOx film, the gel quality and performance improved significantly. It was also noted that the oxygen permeability and transmission of the surface in contact with the polymerising solution was an important factor for consideration. This observation is related to an earlier patent specification (WO 90/13020) in which the oxygen permeability of various plastics (PMMA, PET, polystyrene, polycarbonate and polyethylene) and the implication of oxygen in the plastic is discussed.
The present inventors have now obtained improved electrophoresis gels without the aid of barrier films or chemical treatments. Furthermore, large batch production of gels has been achieved by the use of a new gel-forming apparatus.